Mechanism of FoxR self-cleavage 1 Self-cleavage of the Pseudomonas aeruginosa Cell-Surface Signalling anti-sigma factor FoxR occurs through an N-O acyl rearrangement* Karlijn C. Bastiaansen a,b , Peter van Ulsen b , Maikel Wijtmans c , Wilbert Bitter b , and María A. Llamas a,1 a Department of Environmental Protection, Estación Experimental del Zaidín-Consejo Superior de Investigaciones Científicas, Granada, Spain; b Section of Molecular Microbiology, Department of Molecular Cell Biology and c Division of Medicinal Chemistry, Department of Chemistry and Pharmaceutical Sciences, VU University, Amsterdam, The Netherlands *Running title: Mechanism of FoxR self-cleavage 1 To whom correspondence should be addressed: María A. Llamas, Department of Environmental Protection, Estación Experimental del Zaidín – CSIC, C/ Profesor Albareda 1, 18008 Granada, Spain, Tel.: +34-958181600 Ext. 309 or 291; Fax: +34-958181609; E-mail: marian.llamas@eez.csic.es Keywords: Pseudomonas aeruginosa, siderophore, iron, cell signalling, signal transduction, gene regulation, post- translational modification, proteolysis Background: Many transmembrane anti-sigma factors, including P. aeruginosa FoxR, undergo periplasmic self- cleavage by an unknown mechanism. Results: Presence of an OH or SH group at +1 of the cleavage site is essential for FoxR self-cleavage. Conclusion: FoxR self-cleavage is mediated by an N-O acyl rearrangement. Significance: The complex proteolytic network that modulates alternative sigma factor activity in Gram- negative bacteria also includes an enzyme-independent step. ABSTRACT The Fox system of Pseudomonas aeruginosa is a cell-surface signalling (CSS) 2 pathway employed by the bacterium to sense and respond to the presence of the heterologous siderophore ferrioxamine in the environment. This regulatory pathway controls the transcription of the foxA ferrioxamine receptor gene through the extracytoplasmic function sigma factor  FoxI . In the absence of ferrioxamine the activity of  FoxI is inhibited by the transmembrane anti-sigma factor FoxR. Upon binding of ferrioxamine by the FoxA receptor, FoxR is processed by a complex proteolytic cascade leading to the release and activation of  FoxI . Interestingly, we have recently shown that FoxR undergoes self-cleavage between the periplasmic Gly- 191 and Thr-192 residues independent of the perception of ferrioxamine. This autoproteolytic event, which is widespread among CSS anti-sigma factors, produces two distinct domains that interact and function together to transduce the presence of the signal. In this work, we provide evidence that the self- cleavage of FoxR is not an enzyme-dependent process, but is induced by an N-O acyl rearrangement. Mutation analysis showed that the nucleophilic side chain of the Thr-192 residue at +1 of the cleavage site is required for an attack on the preceding Gly-191 after which the resulting ester bond is likely hydrolysed. Because the cleavage site is well-preserved and the hydrolysis of periplasmic CSS anti-sigma factors is widely observed, we hypothesize that cleavage via an N-O acyl rearrangement is a conserved feature of these proteins. INTRODUCTION The Fox system of the Gram-negative bacterium Pseudomonas aeruginosa is a signal transduction system used by the bacterium to respond to and regulate the uptake of the siderophore ferrioxamine (1). Siderophores are high-affinity iron-chelating compounds that are produced and secreted by bacteria to solubilize the minute amounts of bioavailable iron present in the environment (2,3). P. aeruginosa produces the two siderophores pyoverdine and pyochelin, but is also very efficient in http://www.jbc.org/cgi/doi/10.1074/jbc.M115.643098 The latest version is at JBC Papers in Press. Published on March 25, 2015 as Manuscript M115.643098 Copyright 2015 by The American Society for Biochemistry and Molecular Biology, Inc. at VRIJE UNIVERSITEIT on April 24, 2015 http://www.jbc.org/ Downloaded from